WO1998030693A2 - Recepteur 5 du facteur de necrose tumorale - Google Patents

Recepteur 5 du facteur de necrose tumorale Download PDF

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WO1998030693A2
WO1998030693A2 PCT/US1998/000152 US9800152W WO9830693A2 WO 1998030693 A2 WO1998030693 A2 WO 1998030693A2 US 9800152 W US9800152 W US 9800152W WO 9830693 A2 WO9830693 A2 WO 9830693A2
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thr
pro
polypeptide
ser
cys
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PCT/US1998/000152
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WO1998030693A9 (fr
WO1998030693A3 (fr
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Ying-Fei Wei
Jian Ni
Reinhard Ebner
Guo-Liang Yu
Steven M. Ruben
Reiner L. Gentz
Ping Feng
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Human Genome Sciences, Inc.
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Priority to AU62386/98A priority Critical patent/AU6238698A/en
Priority to JP53103698A priority patent/JP2001505060A/ja
Priority to DE69834027T priority patent/DE69834027D1/de
Priority to EP98904528A priority patent/EP0990031B1/fr
Publication of WO1998030693A2 publication Critical patent/WO1998030693A2/fr
Publication of WO1998030693A3 publication Critical patent/WO1998030693A3/fr
Publication of WO1998030693A9 publication Critical patent/WO1998030693A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention relates to a novel human gene encoding a polypeptide which is a member of the TNF receptor family, and has now been found to bind TRAIL. More specifically, an isolated nucleic acid molecule is provided encoding a human polypeptide named tumor necrosis factor receptor-5, sometimes referred to as "TNFR-5" or “TR5,” and now referred to hereinafter as "TRAIL receptor without intracellular domain” or “TRID.” TRLD polypeptides are also provided, as are vectors, host cells, and recombinant methods for producing the same. The invention further relates to screening methods for identifying agonists or antagonists of TRLD polypeptide activity. Also provided are diagnostic and therapeutic methods utilizing such compositions.
  • cytokines Many biological actions, for instance, response to certain stimuli and natural biological processes, are controlled by factors, such as cytokines. Many cytokines act through receptors by engaging the receptor and producing an intra-cellular response.
  • tumor necrosis factors (TNF) alpha and beta are cytokines, which act through TNF receptors to regulate numerous biological processes, including protection against infection and induction of shock and inflammatory disease.
  • the TNF molecules belong to the "TNF-ligand” superfamily, and act together with their receptors or counter-ligands, the "TNF- receptor” superfamily. So far, nine members of the TNF ligand superfamily have been identified and ten members of the TNF-receptor superfamily have been characterized.
  • TNF- lymphotoxin- ⁇
  • LT- ⁇ lymphotoxin- ⁇
  • LT- ⁇ lymphotoxin- ⁇
  • FasL CD40L, CD27L. CD30L. 4- IBBL, OX40L and nerve growth factor (NGF).
  • the superfamily of TNF receptors includes the p55TNF receptor, p75TNF receptor, TNF receptor-related protein, FAS antigen or APO- 1 , CD40, CD27, CD30, 4-1BB, OX40, low affinity p75 and NGF-receptor (Meager. A., Biologicals 22:291-295 (1994)).
  • TNF-ligand superfamily Many members of the TNF-ligand superfamily are expressed by activated T-cells, implying that they are necessary for T-cell interactions with other cell types which underlie cell ontogeny and functions. (Meager, A., supra).
  • TNF and LT- ⁇ are capable of binding to two TNF receptors (the 55- and 75-kd TNF receptors).
  • TNF and LT- ⁇ are involved in the pathogenesis of a wide range of diseases, including endotoxic shock, cerebral malaria, tumors, autoimmune disease, AIDS and graft-host rejection (Beutler, B. and Von Huffel, C, Science 264:667-668 (1994)). Mutations in the p55 Receptor cause increased susceptibility to microbial infection. Moreover, an about 80 amino acid domain near the C-terminus of TNFR1 (p55) and
  • Fas was reported as the "death domain,” which is responsible for transducing signals for programmed cell death (Tartaglia et al, Cell 74:845 (1993)).
  • Apoptosis or programmed cell death, is a physiologic process essential for the normal development and homeostasis of multicellular organisms (H. whilr, Science 267, 1445-1449 (1995)). Derangements of apoptosis contribute to the pathogenesis of several human diseases including cancer, neurodegenerative disorders, and acquired immune deficiency syndrome (C.B. Thompson, Science 267, 1456-1462 (1995)).
  • One mechanism of immune mediated killing is the engagement of death receptors. Recently, much attention has focused on the signal transduction and biological function of two cell surface death receptors, Fas/APO-1 and TNFR-1 (J.L. Cleveland et al, Cell 81 , 479-482 (1995); A.
  • Fas/APO-1 and TNFR-1 While family members are defined by the presence of cysteine-rich repeats in their extracellular domains, Fas/APO-1 and TNFR-1 also share a region of intracellular homology, appropriately designated the "death domain", which is distantly related to the Drosophila suicide gene, reaper (P. Golstein, et al, Cell 81, 185-186 (1995); K. White et al, Science 264, 677-83 (1994)). This shared death domain suggests that both receptors interact with a related set of signal transducing molecules that, until recently, remained unidentified. Activation of Fas/APO-1 recruits the death domain-containing adapter molecule FADD/MORT1 (A.M.
  • TNFR-1 can signal an array of diverse biological activities-many of which stem from its ability to activate NF-kB (L.A. Tartaglia et al, Immunol Today 13, 151-3 (1992)). Accordingly, TNFR-1 recruits the multivalent adapter molecule TRADD, which like FADD, also contains a death domain (H. Hsu et al, Cell 81, 495-504 (1995); H. Hsu. et al, Cell 84, 299-308 (1996)). Through its associations with a number of signaling molecules including FADD, TRAF2, and RIP, TRADD can signal both apoptosis and NF-kB activation (H. Hsu et al, Cell 84, 299-308 (1996); H. Hsu, et al, Immunity 4, 387- 396 (1996)).
  • TRAIL acts independently from the Fas ligand (Wiley et al, supra). It has also been shown that TRAIL activates apoptosis rapidly, within a time frame that is similar to death signalling by Fas/Apo- 1L, but much faster than TNF-induced apoptosis. S.A. Marsters et al, Current Biology 6, 750-752 (1996). The inability of TRAIL to bind TNFR-1. Fas, or the recently identified DR3, suggests that TRAIL may interact with a unique receptor(s).
  • TNF family ligands and TNF family receptors are varied and influence numerous functions, both normal and abnormal, in the biological processes of the mammalian system. There is a clear need, therefore, for identification and characterization of such receptors and ligands that influence biological activity, both normally and in disease states. In particular, there is a need to isolate and characterize additional novel receptors that bind TRAIL. Summary of the Invention
  • the present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding the TRID polypeptide having the amino acid sequence shown in SEQ LD NO:2, or the amino acid sequence encoded by the cDNA clone deposited as ATCC Deposit Number 97798 on November 20, 1996.
  • the nucleotide sequence determined by sequencing the deposited TRID clone, which is shown in SEQ JD NO: l contains an open reading frame encoding a polypeptide of about 259 amino acid residues, with a leader sequence of about 26 amino acids.
  • the present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells and methods for using them for production of TRID polypeptides or peptides by recombinant techniques.
  • the invention further provides an isolated TRID polypeptide having an amino acid sequence encoded by a polynucleotide described herein.
  • the present invention also provides diagnostic assays such as quantitative and diagnostic assays for detecting levels of TRID protein.
  • a diagnostic assay in accordance with the invention for detecting expression of TRID, or soluble form thereof may be used to detect the ability of normal tissue to withstand or be protected from the deleterious effects of TRAIL, such as TRALL-induced apoptosis.
  • Tumor Necrosis Factor (TNF) family ligands are known to be among the most pleiotropic cytokines, inducing a large number of cellular responses, including cytotoxicity, anti-viral activity, immunoregulatory activities, and the transcriptional regulation of several genes.
  • Cellular response to TNF- family ligands include not only normal physiological responses, but also diseases associated with increased apoptosis or the inhibition of apoptosis.
  • Apoptosis - programmed cell death - is a physiological mechanism involved in the deletion of peripheral T lymphocytes of the immune system, and its dysregulation can lead to a number of different pathogenic processes.
  • Diseases associated with increased cell survival, or the inhibition of apoptosis include cancers, autoimmune disorders, viral infections, inflammation, graft vs. host disease, acute graft rejection, and chronic graft rejection.
  • Diseases associated with increased apoptosis include AIDS, neurodegeneradve disorders, myelodysplastic syndromes, ischemic injury, toxin-induced liver disease, septic shock, cachexia and anorexia.
  • the invention further provides a method for enhancing apoptosis induced by a TNF-family ligand, such as TRAIL, which involves administering to a cell which expresses the TRID polypeptide an effective amount of an antagonist capable of decreasing TRID's ability to bind TRAIL.
  • a TNF-family ligand such as TRAIL
  • TRID binding is decreased to treat a disease wherein decreased apoptosis is exhibited.
  • the present invention is directed to a method for enhancing apoptosis induced by a TNF-family ligand, such as TRAIL, which involves administering to a cell an effective amount of TRID or an agonist capable of increasing TRLD activity.
  • a TNF-family ligand such as TRAIL
  • TRID activity is increased to treat a disease wherein decreased apoptosis is exhibited.
  • Whether any candidate "agonist” or “antagonist” of the present invention can enhance or inhibit apoptosis can be determined using art-known TNF-family ligand/receptor cellular response assays, including those described in more detail below.
  • a screening method is provided for determining whether a candidate agonist or antagonist is capable of enhancing or inhibiting a cellular response to a TNF-family ligand, such as TRAIL.
  • the method involves contacting cells which co-expresses the TRLD polypeptide and a second TNFR with a candidate compound and a TNF-family ligand (e.g., TRAIL), assaying a cellular response, and comparing the cellular response to a standard cellular response, the standard being assayed when contact is made with the ligand in absence of the candidate compound, whereby an increased cellular response over the standard indicates that the candidate compound is a TRID antagonist and a decreased cellular response compared to the standard indicates that the candidate compound is TRLD agonist.
  • a cell expressing the TNFR polypeptide can be contacted with either an endogenous or exo enously administered TNF- family ligand. such as TRAIL.
  • Figure 1 shows the nucleotide sequence (SEQ ID NO:l) and deduced amino acid sequence (SEQ ID NO:2) of TRID.
  • Figure 2 shows an alignment created by the Clustal method using the Megaline program in the DNAstar suite comparing the amino acid sequences of TNFR-5 (now called "TRID”). with other TNF receptors, as follows: TNFR1 (SEQ ID NO:3); TNFR2 (SEQ ID NO:4); NGFR (SEQ ID NO:5) LTbR (SEQ ID NO:6); FAS (SEQ ID NO:7); CD27 (SEQ ID NO:8); CD30 (SEQ LD NO:9); CD40 (SEQ ID NO:10); 4-lBB (SEQ ID NO.T 1); OX40 (SEQ ID NO: 12): VC22 (SEQ ID NO: 13); and CRMB (SEQ ID NO: 14).
  • TNF receptors as follows: TNFR1 (SEQ ID NO:3); TNFR2 (SEQ ID NO:4); NGFR (SEQ ID NO:5) LTbR (SEQ ID NO:6); FAS (SEQ ID NO:7); CD27 (S
  • Figure 3 shows an analyses of the TRID amino acid sequences. Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown.
  • Antigenic Index - Jameson-Wolf graphs the indicate location of the highly antigenic regions of the proteins, i.e., regions from which epitope-bearing peptides of the invention may be obtained.
  • Figure 4 shows the nucleotide sequence of gene fragments related to the TRID gene of the present invention, including: HPRCB54R (SEQ ID NO: 15), HSJAU57RA (SEQ ID NO: 16), HELBP70R (SEQ ID NO: 17), and HUSCB54R (SEQ ID NO: 18) all of which are related to SEQ LD NO: 1.
  • Figure 5A is an immunoblot showing that TRID-Fc (as well as DR4 and DR5) specifically bound TRAIL, but not the related cytotoxic ligand TNF ⁇ .
  • the bottom panel of Fig. 5A shows the input Fc-fusions present in the binding assays.
  • Figure 5B is a bar graph showing that TRID-Fc blocked the ability of TRAIL to induce apoptosis.
  • Figure 5 C is a bar graph showing that TRID-Fc had no effect on TNF ⁇ -induced apoptosis under conditions where TNFRl-Fc completely abolished TNF ⁇ killing.
  • Figure 6 is a bar graph showing that MCF7 cells expressing TRID were protected from TRALL-induced apoptosis, as were cells expressing the virally encoded caspase inhibitor CrmA.
  • the present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding a TRLD polypeptide, having the amino acid sequence shown in Figure 1 (SEQ ID NO:2), which was determined by sequencing a cloned cDNA.
  • the nucleotide sequence shown in Figure 1 (SEQ ID NO:l) was obtained by sequencing the HPRCB54 clone, which was deposited on November 20, 1996 at the American Type Culture Collection, 12301 Park Lawn Drive, Rockville, Maryland 20852, and given accession number ATCC 97798.
  • the deposited clone is inserted in the pBluescript SK(-) plasmid (Stratagene, La Jolla, CA).
  • the TRLD protein of the present invention has an amino acid sequence which is 21.7% identical to and shares multiple conserved cysteine rich domains with the translation product of the human nerve growth factor (hNGF) mRNA (SEQ ID NO:5) as illustrated in Figure 2.
  • hNGF human nerve growth factor
  • SEQ ID NO:5 the translation product of the human nerve growth factor (hNGF) mRNA
  • hNGF is thought to play an important role in the development, survival, apoptosis and function of neurons (Lee. F.K. et al., Cell 69:737) and lymphocytes (Torcia, M. et al, Cell 85:3369 (1996)).
  • TRID extracellular cysteine-rich domain to be strikingly similar to the corresponding domains of both DR4 and DR5 with 69% and 52% amino acid identity, respectively.
  • TRLD was also found to be homologous to the cysteine-rich domain in CAR1, a chicken TNF receptor family member with amino acid identities ranging from 42-48% (J. Brojatsh et al., Cell 87: 1 (1996).
  • a potential protective role for TRID was suggested by the finding that its transcript was detectable in many normal human tissues but not in most transformed cell lines.
  • TRLD has an extracellular TRAIL binding domain and a transmembrane domain but, surprisingly, lacks a putative intracellular signalling domain, in keeping with the possibility that this receptor does not signal following ligand binding. Given the absence of an intracellular domain, this receptor was termed "TRID" for TRAIL Receptor Without an Intracellular Domain.
  • nucleotide sequences determined by sequencing a DNA molecule herein were determined using an automated DNA sequencer (such as the Model 373 from Applied Biosystems, Inc., Foster City, CA), and all amino acid sequences of polypeptides encoded by DNA molecules determined herein were predicted by translation of a DNA sequence determined as above. Therefore, as is known in the art for any DNA sequence determined by this automated approach, any nucleotide sequence determined herein may contain some errors. Nucleotide sequences determined by automation are typically at least about 90% identical, more typically at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule. The actual sequence can be more precisely determined by other approaches including manual DNA sequencing methods well known in the art.
  • a single insertion or deletion in a determined nucleotide sequence compared to the actual sequence will cause a frame shift in translation of the nucleotide sequence such that the predicted amino acid sequence encoded by a determined nucleotide sequence will be completely different from the amino acid sequence actually encoded by the sequenced DNA molecule, beginning at the point of such an insertion or deletion.
  • nucleotide sequence of a nucleic acid molecule or polynucleotide is intended, for a DNA molecule or polynucleotide, a sequence of deoxyribonucleotides, and for an RNA molecule or polynucleotide, the corresponding sequence of ribonucleotides (A, G, C and U), where each thymidine deoxyribonucleotide (T) in the specified deoxyribonucleotide sequence is replaced by the ribonucleotide uridine (U).
  • a nucleic acid molecule of the present invention encoding a TRID polypeptide may be obtained using standard cloning and screening procedures, such as those for cloning cDNAs using mRNA as starting material.
  • the TRID nucleic acid molecule described in SEQ ID NO: l was discovered in a cDNA library derived from prostate tissue. Additional clones of the same gene were also identified in cDNA libraries from the following tissues: endothelial cells, stimulated monocytes, and kerotinocytes.
  • the determined nucleotide sequence of the TRID cDNA of SEQ ID NO: 1 contains an open reading frame encoding a protein of about 259 amino acid residues, with an initiation codon at nucleotide positions 183-185 of the nucleotide sequences in Figure 1 (SEQ ID NO: l).
  • the open reading frame of the TRID gene shares sequence homology with the translation product of the human mRNA for NGFR. including the following conserved domains: (a) a soluble extracellular domain of about 214 amino acids (residues 27-240 of Figure 1); and (b) a transmembrane domain of about 19 amino acids (residues 241-259 of Figure 1).
  • the actual complete TRLD polypeptide encoded by the deposited cDNAs which comprise about 259 amino acids, may be somewhat longer or shorter. More generally,
  • the actual open reading frames may be anywhere in the range of ⁇ 20 amino acids, more likely in the range of ⁇ 10 amino acids, of that predicted from the first methionine codon from the N- terminus shown in SEQ ID NO: 1 , which is in-frame with the translated sequences shown in each respective figure. It will further be appreciated that, depending on the analytical criteria used for identifying various functional domains, the exact "address" of the extracellular and transmembrane domain(s) of the TNFR polypeptides may differ slightly from the predicted positions above.
  • the exact location of the extracellular domain in SEQ ID NO:2 may vary slightly (e.g., the address may "shift" by about 1 to about 20 residues, more likely about 1 to about 5 residues) depending on the criteria used to define the domain.
  • the beginning of the transmembrane domain and the end of the extracellular domain were predicted on the basis of the identification of the hydrophobic amino acid sequence in the above indicated positions, as shown in Figure 5.
  • the invention further provides polypeptides having various residues deleted from the N-terminus of the complete polypeptide, including polypeptides lacking one or more amino acids from the N-terminus of the extracellular domain described herein, which constitute soluble forms of the extracellular domain of the TRID protein.
  • the amino acid sequence of the TRLD protein includes a leader sequence and a mature protein, as shown in SEQ ID NO:2. More in particular, the present invention provides nucleic acid molecules encoding mature forms of the TRID protein.
  • proteins secreted by mammaHan cells have a signal or secretory leader sequence which is cleaved from the complete polypeptide to produce a secreted "mature" form of the protein.
  • Most mammalian cells and even insect cells cleave secreted proteins with the same specificity. However, in some cases, cleavage of a secreted protein is not entirely uniform, which results in two or more mature species of the protein.
  • the present invention provides a nucleotide sequence encoding a mature TRLD polypeptide having the amino acid sequence encoded by a cDNA clone identified as ATCC Deposit No. 97798.
  • a mature TRLD polypeptide having the amino acid sequence encoded by a cDNA clone in ATCC Deposit No. 97798 * is meant the mature form(s) of the protein produced by expression in a mammalian cell (e.g.. COS cells, as described below) of the complete open reading frame encoded by the human DNA sequence of the clone contained in the deposited plasmid.
  • Virus Res. 3:271-286 (1985) uses the information from a short N-terminal charged region and a subsequent uncharged region of the complete (uncleaved) protein.
  • the method of von Heinje uses the information from the residues surrounding the cleavage site, typically residues -13 to +2 where +1 indicates the amino terminus of the mature protein.
  • the accuracy of predicting the cleavage points of known mammalian secretory proteins for each of these methods is in the range of 75-80% (von Heinje, supra). However, the two methods do not always produce the same predicted cleavage point(s) for a given protein.
  • PSORT is an expert system for predicting the cellular location of a protein based on the amino acid sequence.
  • the analysis by the PSORT program predicted the cleavage sites between amino acids -1 and 1 in SEQ ID NO:2. Thereafter, the complete amino acid sequences were further analyzed by visual inspection, applying a simple form of the (-1,-3) rule of von Heinje. von Heinje, supra.
  • the leader sequence for the TRID protein is predicted to consist of amino acid residues from about 1 to about 26, underlined in Figure 1 (corresponding to about -26 to about -1 in SEQ ID NO:2), while the mature TRID protein is predicted to consist of residues from about 1 to about 233 in SEQ ID NO:2.
  • the mature TRLD polypeptide encoded by the deposited cDNA comprises about 233 amino acids, but may be anywhere in the range of about 223 to about 243 amino acids, and the predicted leader sequence of this protein is about 26 amino acids, but may be anywhere in the range of about 16 to about 36 amino acids.
  • nucleic acid molecules of the present invention may be in the form of RNA, such as mRNA, or in the form of DNA, including, for instance, cDNA and genomic DNA obtained by cloning or produced synthetically.
  • the DNA may be double-stranded or single-stranded.
  • Single-stranded DNA or RNA may be the coding strand, also known as the sense strand, or it may be the non-coding strand, also referred to as the anti-sense strand.
  • isolated nucleic acid molecule(s) is intended a nucleic acid molecule, DNA. or RNA, which has been removed from its native environment.
  • recombinant DNA molecules contained in a vector are considered isolated for the purposes of the present invention.
  • Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution.
  • Isolated RNA molecules include in vivo or in vitro RNA transcripts of the DNA molecules of the present invention. Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically.
  • Isolated nucleic acid molecules of the present invention include DNA molecules comprising an open reading frame (ORF) shown in SEQ ID NO:l; DNA molecules comprising the coding sequence for the mature TRID protein; and DNA molecules which comprise a sequence substantially different from those described above, but which, due to the degeneracy of the genetic code, still encode the TRLD protein.
  • ORF open reading frame
  • DNA molecules comprising the coding sequence for the mature TRID protein
  • ORF open reading frame
  • the invention provides nucleic acid molecules having nucleotide sequences related to extensive portions of SEQ ID NO: l, which have been determined from the following related cDNA clones: HELBP70R (SEQ LD NO: 17), HPRCB54R (SEQ LD NO: 15), HSJAU57RA (SEQ ID NO:16) and HUSCB54R (SEQ LD NO:18).
  • HELBP70R SEQ LD NO: 17
  • HPRCB54R SEQ LD NO: 15
  • HSJAU57RA SEQ ID NO:16
  • HUSCB54R SEQ LD NO:18
  • the nucleotide sequences of each of these gene fragments is shown in Figure 4.
  • the invention provides isolated nucleic acid molecules encoding the
  • TRLD polypeptide having an amino acid sequence as encoded by the cDNA clone contained in the plasmid deposited as ATCC Deposit No. 97798.
  • nucleic acid molecules are provided that encode the mature TRID polypeptide or the full length TRLD polypeptide each lacking the N-terminal methionine.
  • the invention further provides an isolated nucleic acid molecule having the nucleotide sequence shown in SEQ LD NO: 1 or the nucleotide sequence of the TRID cDNA contained in the above-described deposited clone, or a nucleic acid molecule having a sequence complementary to one of the above sequences.
  • Such isolated molecules, particularly DNA molecules are useful as probes for gene mapping, by in situ hybridization with chromosomes, and for detecting expression of the TRID gene in human tissue, for instance, by Northern blot analysis.
  • the present invention is further directed to fragments of the isolated nucleic acid molecules described herein.
  • a fragment of an isolated nucleic acid molecule having the nucleotide sequence of the deposited cDNA or the nucleotide sequence shown in SEQ LD NO: 1 is intended fragments at least about 15 nt. and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt in length which are useful as diagnostic probes and primers as discussed herein.
  • fragments 50-300 nt, or even 600 nt in length are also useful according to the present invention as are fragments corresponding to most, if not all, of the nucleotide sequence of the deposited cDNA or as shown in SEQ ID NO:l.
  • a fragment at least 20 nt in length for example, is intended fragments which include 20 or more contiguous bases from the nucleotide sequence of a deposited cDNA or the nucleotide sequence as shown in SEQ ID NO:l.
  • Preferred nucleic acid fragments of the present invention include nucleic acid molecules encoding: epitope-bearing portions of the TRLD polypeptide as identified in Figure 3 and described in more detail below.
  • the invention provides polynucleotides having a nucleotide sequence representing the portion of SEQ LD NO: l, which consist of positions 183-959 of SEQ ID NO: l. Also contemplated are polynucleotides encoding TRID polypeptides which lack an amino terminal methionine.
  • One such preferred polynucleotide has a nucleotide sequence representing the portion of SEQ LD NO:l which consists of positions 186-959.
  • Polypeptides encoded by such polynucleotides are also provided, such polypeptides comprising an amino acid sequence at positions 2-259 of SEQ ID NO: 2, or the polypeptide sequence encoded by the clone deposited with the ATCC as Deposit No. 97798 lacking an amino terminal methionine.
  • Preferred nucleic acid fragments of the present invention include nucleic acid molecules encoding: a polypeptide comprising the TRLD extracellular domain (amino acid residues from about 27 to about 240 in FIG. 1 (from about 1 to about 214 in SEQ LD NO:2)); and a polypeptide comprising the TRID transmembrane domain (amino acid residues from about 241 to about 259 in FIG. 1 (from about 215 to about 233 in SEQ ID NO:2)). Since the location of these domains have been predicted by computer graphics, one of ordinary skill would appreciate that the amino acid residues constituting these domains may vary slightly (e.g., by about 1 to 15 residues) depending on the criteria used to define each domain.
  • Preferred nucleic acid fragments of the invention encode a full-length
  • TRLD polypeptide lacking the nucleotides encoding the amino-terminal methionine (e.g., nucleotides 186-959 in SEQ LD NO: l) as it is known that the methionine is cleaved naturally and such sequences maybe useful in genetically engineering TRLD expression vectors.
  • Polypeptides encoded by such polynucleotides are also contemplated by the invention.
  • nucleic acid fragments of the present invention further include nucleic acid molecules encoding epitope-bearing portions of the TRLD protein.
  • nucleic acid fragments of the present invention include nucleic acid molecules encoding: a polypeptide comprising amino acid residues from about Gln-42 to about Glu-52 in Figure 1 (about Gin- 16 to about Glu-26 in SEQ ID NO:2); a polypeptide comprising amino acid residues from about His-58 to about Cys-66 in Figure 1 (about His-32 to about Cys-40 in SEQ ID NO:2); a polypeptide comprising amino acid residues from about Pro-68 to about Thr-76 in Figure 1 (about Pro-42 to about Thr-50 in SEQ ID NO:2); a polypeptide comprising amino acid residues from about Ser-79 to about Cys-85 in Figure 1 (about Ser-53 to about Cys-59 in SEQ LD NO:2); a polypeptide comprising amino acid residues from about Cys-91 to
  • the invention provides an isolated nucleic acid molecule comprising a polynucleotide which hybridizes under stringent hybridization conditions to a portion of the polynucleotide in a nucleic acid molecule of the invention described above, for instance, a cDNA clone contained in ATCC Deposit No. 97798.
  • stringent hybridization conditions is intended overnight incubation at 42° C in a solution comprising: 50% formamide, 5x SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 ⁇ g/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0. lx SSC at about 65° C.
  • a polynucleotide which hybridizes to a "portion" of a polynucleotide is intended a polynucleotide (either DNA or RNA) hybridizing to at least about 15 nucleotides (nt), and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably about 30-70 (e.g., 50) nt of the reference polynucleotide. These are useful as diagnostic probes and primers as discussed above and in more detail below.
  • a polynucleotide which hybridizes only to a poly A sequence such as the 3 ' terminal ⁇ oly(A) tract of the TRLD cDNA shown in SEQ ID NO:l), or to a complementary stretch of T (or U) residues, would not be included in a polynucleotide of the invention used to hybridize to a portion of a nucleic acid of the invention, since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any double-stranded cDNA clone).
  • nucleic acid molecules of the present invention which encode a TRLD polypeptide may include, but are not limited to the coding sequence for the mature polypeptide, by itself; the coding sequence for the mature polypeptide and additional sequences, such as those encoding a leader or secretary sequence, such as a pre-, or pro- or prepro- protein sequence; the coding sequence of the mature polypeptide, with or without the aforementioned additional coding sequences, together with additional, non-coding sequences, including for example, but not limited to introns and non-coding 5 1 and 3' sequences, such as the transcribed, non-translated sequences that play a role in transcription, mRNA processing - including splicing and polyadenylation signals, for example - ribosome binding and stability of mRNA; additional coding sequence which codes for additional amino acids, such as those which provide additional functionalities.
  • the polypeptide may be fused to a marker sequence, such as a peptide, which facilitates purification of the
  • the marker sequence is a hexa- histidine peptide, such as the tag provided in a pQE vector (Qiagen, Inc.), among others, many of which are commercially available.
  • a pQE vector Qiagen, Inc.
  • hexa-histidine provides for convenient purification of the fusion protein.
  • the "HA" tag is another peptide useful for purification which corresponds to an epitope derived from the influenza hemagglutinin protein, which has been described by Wilson et al, Cell 37:167 -778(1984).
  • other such fusion proteins include the TRID receptor fused to Fc at the N- or C- terminus.
  • the present invention further relates to variants of the nucleic acid molecules of the present invention, which encode portions, analogs, or derivatives of the TRLD receptor.
  • Variants may occur naturally, such as a natural allelic variant.
  • allelic variant is intended one of several alternate forms of a gene occupying a given locus on a chromosome of an organism. Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985). Non-naturally occurring variants may be produced using art-known mutagenesis techniques.
  • variants include those produced by nucleotide substitutions, deletions or additions.
  • the substitutions, deletions or additions may involve one or more nucleotides.
  • the variants may be altered in coding regions, non-coding regions, or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions. Especially preferred among these are silent substitutions, additions and deletions, which do not alter the properties and activities of the TRID polypeptide or portions thereof. Also especially preferred in this regard are conservative substitutions.
  • nucleic acid molecule comprising a polynucleotide having a nucleotide sequence at least 90% identical, and more preferably at least 95%, 96%, 97%, 98% or 99% identical to: (a) a nucleotide sequence encoding the polypeptide having the amino acid sequence in SEQ LD NO:2; (b) a nucleotide sequence encoding the polypeptide having the amino acid sequence in SEQ LD NO:2, but lacking the amino terminal methionine; (c) a nucleotide sequence encoding the polypeptide having the amino acid sequence at positions about 1 to about 233 in SEQ LD NO:2; (d) a nucleotide sequence encoding the polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No.
  • inventions include isolated nucleic acid molecules that comprise a polynucleotide which hybridizes under stringent hybridization conditions to a polynucleotide in (a), (b), (c), (d), (e), (f), (g) or (h) above.
  • This polynucleotide which hybridizes does not hybridize under stringent hybridization conditions to a polynucleotide having a nucleotide sequence consisting of only A residues or of only T residues.
  • An additional nucleic acid embodiment of the invention relates to an isolated nucleic acid molecule comprising a polynucleotide which encodes the amino acid sequence of an epitope-bearing portion of a TRLD polypeptide having an amino acid sequence in (a), (b), (c), (d), (e), (f) or (g) above.
  • a polynucleotide having a nucleotide sequence at least, for example, 95% "identical" to a reference nucleotide sequence encoding a TRID polypeptide is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence encoding the TRLD polypeptide.
  • a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence.
  • These mutations of the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
  • nucleic acid molecule is at least 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the nucleotide sequence shown in SEQ ID NO:l, or to the nucleotide sequence of the deposited cDNA clone can be determined conventionally using known computer programs such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park. 575 Science Drive, Madison, WI 53711). Bestfit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981). to find the best segment of homology between two sequences.
  • Bestfit program Wiconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park. 575 Science Drive, Madison, WI 53711. Bestfit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981). to find the best segment of homology between two sequences.
  • the parameters are set. of course, such that the percentage of identity is calculated over the full length of the reference nucleotide sequence and that gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed.
  • the present application is directed to nucleic acid molecules at least 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleic acid sequence shown in SEQ LD NO: l, or to the nucleic acid sequence of the deposited cDNA, irrespective of whether they encode a polypeptide having TRLD activity. This is because even where a particular nucleic acid molecule does not encode a polypeptide having TRID activity, one of skill in the art would still know how to use the nucleic acid molecule, for instance, as a hybridization probe or a polymerase chain reaction (PCR) primer.
  • PCR polymerase chain reaction
  • nucleic acid molecules of the present invention that do not encode a polypeptide having TRLD activity include, inter alia: (1) isolating a TRLD gene or allelic variants thereof in a cDNA library; (2) in situ hybridization (e.g., "FISH") to metaphase chromosomal spreads to provide precise chromosomal location of the TRLD gene, as described in Verma et al., Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York (1988); and Northern Blot analysis for detecting TRID mRNA expression in specific tissues.
  • FISH in situ hybridization
  • nucleic acid molecules having sequences at least 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleic acid sequence shown in SEQ ID NO: 1, or to the nucleic acid sequence of the deposited cDNA which does, in fact, encode a polypeptide having TRLD receptor activity.
  • a polypeptide having TRLD receptor activity is intended polypeptides exhibiting activity similar, but not necessarily identical, to an activity of the TRLD receptor of the invention (either the full length protein or preferably the mature protein or extracellular domain alone), as measured in a particular biological assay.
  • the TNF family ligands include TRAIL
  • a cellular response to a TNF-family ligand is intended any genotypic, phenotypic, and/or morphological change to a cell, cell line, tissue, tissue culture or patient that is induced by a TNF-family ligand.
  • cellular responses include not only normal physiological responses to TNF- family ligands, but also diseases associated with increased cell proliferation or the inhibition of increased cell proliferation, such as by the inhibition of apoptosis.
  • nucleic acid molecules having a sequence at least 90%. 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of a deposited cDNA or the nucleic acid sequence shown in SEQ LD NO: 1 will encode a polypeptide "having TRLD protein activity.”
  • degenerate variants of these nucleotide sequences all encode the same polypeptide, this will be clear to the skilled artisan even without performing the above described comparison assay. It will be further recognized in the art that, for such nucleic acid molecules that are not degenerate variants, a reasonable number will also encode a polypeptide "having TRLD protein activity."
  • polypeptide having TRID protein activity This is because the skilled artisan is fully aware of amino acid substitutions that are either less likely or not likely to significantly effect protein function (e.g., replacing one aliphatic amino acid with a second aliphatic amino acid), as further described below.
  • the present invention also relates to vectors which include the isolated DNA molecules of the present invention, host cells which are genetically engineered with the recombinant vectors of the invention and the production of TRLD polypeptides or fragments thereof by recombinant techniques.
  • the polynucleotides may be joined to a vector containing a selectable marker for propagation in a host.
  • a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.
  • the DNA insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few.
  • an appropriate promoter such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few.
  • Other suitable promoters will be known to the skilled artisan.
  • the expression constructs will further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation.
  • the coding portion of the mature transcripts expressed by the constructs will preferably include a translation initiating at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.
  • the expression vectors will preferably include at least one selectable marker.
  • markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture and tetracycline or ampicillin resistance genes for culturing in E. coli and other bacteria.
  • Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS and Bowes melanoma cells; and plant cells. Appropriate culture mediums and conditions for the above-described host cells are known in the art.
  • vectors preferred for use in bacteria include pQ ⁇ 70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRLT5 available from Pharmacia.
  • preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia.
  • the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al, Basic Methods In Molecular Biology (1986).
  • the polypeptide may be expressed in a modified form, such as a fusion protein, and may include not only secretion signals but also additional heterologous functional regions. Thus, for instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification or during subsequent handling and storage.
  • peptide moieties may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide.
  • a preferred fusion protein comprises a heterologous region from immunoglobulin that is useful to solubilize proteins.
  • EP-A-O 464 533 (Canadian counterpart 2045869) discloses fusion proteins comprising various portions of constant region of immunoglobin molecules together with another human protein or part thereof.
  • the Fc part in a fusion protein is thoroughly advantageous for use in therapy and diagnosis and thus results, for example, in improved pharmacokinetic properties (EP-A 0232 262).
  • human proteins such as the hLL5-receptor, have been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5. See, D. Bennett et al, Journal of Molecular Recognition 8:52-58 (1995) and K. Johanson et al, The Journal of Biological Chemistry 270:16:9459-9471 (1995).
  • the TRJLD receptor can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography (“HPLC”) is employed for purification.
  • HPLC high performance liquid chromatography
  • Polypeptides of the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells. Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or non-glycosylated. In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.
  • TRLD receptor polynucleotides and polypeptides may be used in accordance with the present invention for a variety of applications, particularly those that make use of the chemical and biological properties of TRID. Among these are applications in treatment of tumors, resistance to parasites, bacteria and viruses, to induce proliferation of T-cells, endothelial cells and certain hematopoietic cells, to treat restenosis, graft vs. host disease, to regulate anti-viral responses and to prevent certain autoimmune diseases after stimulation of TRLD by an agonist.
  • Additional applications relate to diagnosis and to treatment of disorders of cells, tissues and organisms. These aspects of the invention are discussed further below.
  • the invention further provides an isolated TRID polypeptide having the amino acid sequences encoded by the deposited cDNA, or the amino acid sequences in SEQ ID NO:2, or a peptide or polypeptide comprising a portion of the above polypeptides.
  • TRLD polypeptide To improve or alter the characteristics of a TRLD polypeptide, protein engineering may be employed. Recombinant DNA technology known to those skilled in the art can be used to create novel mutant proteins or "muteins" including single or multiple amino acid substitutions, deletions, additions or fusion proteins. Such modified polypeptides can show, e.g., enhanced activity or increased stability. In addition, they may be purified in higher yields and show better solubility than the corresponding natural polypeptide. at least under certain purification and storage conditions.
  • N -Terminal and C-Terminal Deletion Mutants For instance, for many proteins, including the extracellular domain of a membrane associated protein or the mature form(s) of a secreted protein, it is known in the art that one or more amino acids may be deleted from the N-terminus or C-terminus without substantial loss of biological function. For instance, Ron et al, J. Biol. Chem., 2(55:2984-2988 (1993) reported modified KGF proteins that had heparin binding activity even if 3, 8, or 27 amino- terminal amino acid residues were missing.
  • deletions of N-terminal amino acids up to the cysteine at position C-53 of Figure 1 may retain some biological activity such as regulation of proliferation and apoptosis of lymphoid cells.
  • Polypeptides having further N-terminal deletions including the C53 residue in Figure 1 (C-27 in SEQ ID NO:2). would not be expected to retain such biological activities because it is known
  • the present invention further provides polypeptides having one or more residues deleted from the amino terminus of the amino acid sequence shown in Figure 1 , up to the cysteine residue in each which is at position number 53, and polynucleotides encoding such polypeptides.
  • the present invention provides TRLD polypeptides comprising the amino acid sequence of residues m-259 of Figure 1 where m is an integer in the range of 1-53 where 53 is the position of the first cysteine residue from the N-terminus of the complete TRLD polypeptide (shown in Figure 1) believed to be required for activity of the TRID protein.
  • the invention provides polynucleotides encoding polypeptides having the amino acid sequence of residues: 1-259, 2-259, 3-259, 4-259, 5-259, 6-259, 7- 259, 8-259, 9-259, 10-259, 1 1-259, 12-259, 13-259, 14-259, 15-259, 16-259, 17-259, 18- 259, 19-259, 20-259, 21-259, 22-259, 23-259, 24-259, 25-259, 26-259, 27-259, 28-259, 29- 259, 30-259, 31-259, 32-259, 33-259, 34-259, 35-259, 36-259, 37-259, 38-259, 39-259, 40- 259, 41-259, 42-259, 43-259, 44-259, 45-259, 46-259, 47-259. 48-259, 49-259, 50-259. 51- 259, 52-259. and 53-259 of Figure 1. Polynucleotides,
  • C-terminal deletion muteins are known. For instance, interferon gamma shows up to ten times higher activities by deleting 8- 10 amino acid residues from the carboxy terminus of the protein (Dobeli et al.. J. Biotechnology 7:199-216 (1988)).
  • the protein of the invention is a member of the TNFR polypeptide family
  • deletions of C-terminal amino acids up to the cysteine at position 149 of Figure 1 may retain some biological activity such as regulation of proliferation and apoptosis of lymphoid cells.
  • Polypeptides having further C-terminal deletions including the cysteine at position 149 of Figure 1 would not be expected to retain such biological activities because it is known that this residue in TNF receptor-related polypeptides is required for forming a disulfide bridge to provide structural stability which is needed for ligand binding.
  • the present invention further provides polypeptides having one or more residues from the carboxy terminus of the amino acid sequence of TRID shown in Figure 1 up to the cysteine at position 149 of Figure 1, and polynucleotides encoding such polypeptides.
  • the present invention provides polypeptides having the amino acid sequence of residues 1-x of the amino acid sequence in Figure 1, where x is any integer in the range of 149- 259. Polynucleotides encoding these polypeptides also are provided.
  • the invention also provides polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini, which may be described generally as having residues m-x of Figure 1, where m and x are integers as described above. Also included are a nucleotide sequence encoding a polypeptide consisting of a portion of a complete TRLD amino acid sequence encoded by a cDNA clone contained in ATCC Deposit No. 97798, where this portion excludes from 1 to about 49 amino acids from the amino terminus of the complete amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No.
  • the invention further includes variations of the TRLD polypeptide, which show substantial TRLD polypeptide activity or which include regions of TRLD protein such as the protein portions discussed below.
  • Such mutants include deletions, insertions, inversions, repeats, and type substitutions
  • the fragment, derivative, or analog of the polypeptide of SEQ LD NO:2, or that encoded by the deposited cDNA may be: (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue(s), and more preferably at least one but less than ten conserved amino acid residue(s)), and such substituted amino acid residue(s) may or may not be one encoded by the genetic code; or (ii) one in which one or more of the amino acid residues includes a substituent group;or (iii) one in which the mature or soluble extracellular polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol); or (iv) one in which the additional amino acids are fused to the mature polypeptide, such as an IgG Fc fusion region peptide or leader or secretory sequence or a sequence which is employed for purification of
  • the TRID of the present invention may include one or more amino acid substitutions, deletions or additions, either from natural mutations or human manipulation. As indicated, changes are preferably of a minor nature, such as conservative amino acid substitutions that do not significantly affect the folding or activity of the protein (see Table 1).
  • Amino acids in the TRID protein of the present invention that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine- scanning mutagenesis (Cunningham and Wells, Science 244: 1081-1085 (1989)). The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity such as receptor binding or in vitro proliferative activity. Of particular interest are substitutions of charged amino acids with another charged amino acids and with neutral or negatively charged amino acids. The latter results in proteins with reduced positive charge to improve the characteristics of the TRID protein. The prevention of aggregation is highly desirable.
  • the replacement of amino acids can also change the selectivity of binding of a ligand to cell surface receptors.
  • Ostade et al Nature 361:266-268 (1993) describes certain mutations resulting in selective binding of TNF- ⁇ to only one of the two known types of TNF receptors.
  • Sites that are critical for ligand-receptor binding can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith et al, J. Mol. Biol. 224:899-904 (1992) and de Vos et al. Science 255:306-312 (1992)).
  • the polypeptides of the present invention are preferably provided in an isolated form.
  • isolated polypeptide is intended a polypeptide removed from its native environment.
  • a polypeptide produced and/or contained within a recombinant host cell is considered isolated for purposes of the present invention.
  • isolated polypeptide are polypeptides that have been purified, partially or substantially, from a recombinant host cell.
  • a recombinantly produced version of the TRLD polypeptide can be substantially purified by the one-step method described in Smith and Johnson, Gene 67:31-40 (1988).
  • the TRID polypeptide is a 259 residue protein exhibiting two main structural domains.
  • the extracellular TRAIL ligand binding domain was identified within residues from about 1 to about 214 in SEQ LD NO:2.
  • the transmembrane domain was identified within residues from about 215 to about 233 in SEQ LD NO:2.
  • TRID surprisingly lacks a putative intracellular signalling domain, thus, the name 'TRID" (TRAIL Receptor Without an Intracellular Domain").
  • polypeptides of the present invention include the polypeptide encoded by the deposited cDNA including the leader; the mature polypeptide encoded by the deposited the cDNA minus the leader (i.e., the mature protein); a polypeptide comprising amino acids about - 26 to about 233 in SEQ LD NO:2; a polypeptide comprising amino acids about - 25 to about 233 in SEQ LD NO:2; a polypeptide comprising amino acids about 1 to about 233 in SEQ ID NO:2; a polypeptide comprising the extracellular domain; and a polypeptide comprising the transmembrane domain; as well as polypeptides which are at least 80% identical, more preferably at least 90% or 95% identical, still more preferably at least 96%, 97%, 98%, or 99% identical to the polypeptides described above, and also include portions of such polypeptides with at least 30 amino acids and more preferably at least 50 amino acids.
  • polypeptide having an amino acid sequence at least, for example, 95% "identical" to a reference amino acid sequence of a TRID polypeptide is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid of the TRID polypeptide.
  • up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence.
  • alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
  • any particular polypeptide is at least 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the amino acid sequence shown in SEQ LD NO:2, or to the amino acid sequence encoded by the deposited cDNA clone, can be determined conventionally using known computer programs such the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 5371 1).
  • the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference amino acid sequence and that gaps in homology of up to 5% of the total number of amino acid residues in the reference sequence are allowed.
  • polypeptide of the present invention could be used as a molecular weight marker on SDS-PAGE gels or on molecular sieve gel filtration columns using methods well known to those of skill in the art.
  • the invention provides a peptide or polypeptide comprising an epitope-bearing portion of a polypeptide of the invention.
  • the epitope of this polypeptide portion is an immunogenic or antigenic epitope of a polypeptide of the invention.
  • An "immunogenic epitope” is defined as a part of a protein that elicits an antibody response when the whole protein is the immunogen.
  • a region of a protein molecule to which an antibody can bind is defined as an "antigenic epitope.”
  • the number of immunogenic epitopes of a protein generally is less than the number of antigenic epitopes. See, for instance, Geysen et al, Proc. Natl. Acad. Sci.
  • Peptides capable of eliciting protein-reactive sera are frequently represented in the primary sequence of a protein, can be characterized by a set of simple chemical rules, and are confined neither to irnmunodominant regions of intact proteins (i.e., immunogenic epitopes) nor to the amino or carboxyl terminals.
  • Antigenic epitope-bearing peptides and polypeptides of the invention are therefore useful to raise antibodies, including monoclonal antibodies, that bind specifically to a polypeptide of the invention. See, for instance, Wilson et al, Cell 37:767-718 (1984) at 777.
  • Antigenic epitope-bearing peptides and polypeptides of the invention preferably contain a sequence of at least seven, more preferably at least nine and most preferably between about f-Y 15 to about 30 amino acids contained within the amino acid sequence of a polypeptide of the invention.
  • Non-limiting examples of antigemc polypeptides or peptides that can be used to generate TRID-specific antibodies include: a polypeptide comprising amino acid residues from about Gln-42 to about Glu-52 in Figure 1 (about Gin- 16 to about Glu-26 in SEQ ID NO:2); a polypeptide comprising amino acid residues from about His-58 to about Cys-66 in Figure 1 (about His-32 to about Cys-40 in SEQ LD NO:2); a polypeptide comprising amino acid residues from about Pro-68 to about Thr-76 in Figure 1 (about Pro-42 to about Thr-50 in SEQ LD NO:2); a polypeptide comprising amino acid residues from about Ser-79 to about Cys-85 in Figure 1 (about Ser-53 to about Cys-59 in SEQ ID NO:2); a polypeptide comprising amino acid residues from about Cys-91 to about Thr- 102 in Figure 1 (about Cys-65 to about Thr-76 in SEQ ID NO
  • the epitope-bearing peptides and polypeptides of the invention may be produced by any conventional means. See, e.g., Houghten, R. A. (1985) "General method for the rapid solid-phase synthesis of large numbers of peptides: specificity of antigen-antibody interaction at the level of individual amino acids.” Proc. Natl. Acad. Sci. USA 52:5131-5135; this "Simultaneous Multiple Peptide Synthesis (SMPS)" process is further described in U.S. Patent No. 4,631,211 to Houghten et al. (1986). Epitope-bearing peptides and polypeptides of the invention are used to induce antibodies according to methods well known in the art. See.
  • SMPS Simultaneous Multiple Peptide Synthesis
  • Immunogenic epitope-bearing peptides of the invention i.e., those parts of a protein that elicit an antibody response when the whole protein is the immunogen, are identified according to methods known in the art. See, for instance, Geysen et al., supra. Further still, U.S. Patent No.
  • TRLD receptor polypeptides of the present invention and the epitope-bearing fragments thereof described above can be combined with parts of the constant domain of immunoglobulins (IgG), resulting in chimeric polypeptides.
  • IgG immunoglobulins
  • fusion proteins facilitate purification and show an increased half-life in vivo. This has been shown, e.g., for chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins (EP A 394,827; Traunecker et al.. Nature 331:84-86 (1988)).
  • Fusion proteins that have a disulfide-linked dimeric structure due to the IgG part can also be more efficient in binding and neutralizing other molecules than the monomeric TRLD protein or protein fragment alone (Fountoulakis et al, J. Biochem. 270:3958-3964 (1995)).
  • TRLD-protein specific antibodies for use in the present invention can be raised against the intact TRIDproteins or an antigenic polypeptide fragment thereof, which may be presented together with a carrier protein, such as an albumin, to an animal system (such as rabbit or mouse) or, if it is long enough (at least about 25 amino acids), without a carrier.
  • a carrier protein such as an albumin
  • antibody As used herein, the term "antibody” (Ab) or “monoclonal antibody” (Mab) is meant to include intact molecules as well as antibody fragments (such as, for example, Fab and F(ab')2 fragments) which are capable of specifically binding to a TNFR protein.
  • Fab and F(ab')2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding of an intact antibody (Wahl et al, J. Nucl. Med. 24:316-325 (1983)). Thus, these fragments are preferred.
  • the antibodies of the present invention may be prepared by any of a variety of methods.
  • cells expressing the TRID protein or an antigenic fragment thereof can be administered to an animal in order to induce the production of sera containing polyclonal antibodies.
  • a preparation of TRLD protein is prepared and purified to render it substantially free of natural contaminants. Such a preparation is then introduced into an animal in order to produce polyclonal antisera of greater specific activity.
  • the antibodies of the present invention are monoclonal antibodies.
  • Such monoclonal antibodies can be prepared using hybridoma technology (K ⁇ hler et a , Nature 256:495 (1975); K ⁇ hler et al, Eur. J. Immunol. 6:5X 1 (1976); K ⁇ hler et al., Eur. J. Immunol. 6:292 (1976); Hammerling et al, in: Monoclonal Antibodies and T-Cell
  • Such procedures involve immunizing an animal (preferably a mouse) with a TRLD protein antigen or, more preferably, with a TRID protein-expressing cell.
  • Suitable cells can be recognized by their capacity to bind anti-TRID protein antibody.
  • Such cells may be cultured in any suitable tissue culture medium; however, it is preferable to culture cells in Earle's modified Eagle's medium supplemented with 10% fetal bovine serum (inactivated at about 56° C), and supplemented with about 10 g/1 of nonessential amino acids, about 1,000 U/ml of penicillin, and about 100 ⁇ g/ml of streptomycin.
  • the splenocytes of such mice are extracted and fused with a suitable myeloma cell line.
  • a suitable myeloma cell line may be employed in accordance with the present invention; however, it is preferable to employ the parent myeloma cell line (SP2O), available from the American Type Culture Collection, Rockville, Maryland.
  • SP2O parent myeloma cell line
  • the resulting hybridoma cells are selectively maintained in HAT medium, and then cloned by limiting dilution as described by Wands et al. (Gastroenterology 80:225-232 (1981)).
  • the hybridoma cells obtained through such a selection are then assayed to identify clones which secrete antibodies capable of binding the desired TRID antigen.
  • additional antibodies capable of binding to the TRID antigen may be produced in a two-step procedure through the use of anti-idiotypic antibodies.
  • TRID-protein specific antibodies are used to immunize an animal, preferably a mouse.
  • the splenocytes of such an animal are then used to produce hybridoma cells, and the hybridoma cells are screened to identify clones which produce an antibody whose ability to bind to the TRID protein-specific antibody can be blocked by the TRLD protein antigen.
  • Such antibodies comprise anti-idiotypic antibodies to the TRID protein-specific antibody and can be used to immunize an animal to induce formation of further TRID protein-specific antibodies.
  • Fab and F(ab')2 and other fragments of the antibodies of the present invention may be used according to the methods disclosed herein.
  • Such fragments are typically produced by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments).
  • enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments).
  • TRLD protein-binding fragments can be produced through the application of recombinant DNA technology or through synthetic chemistry.
  • chimeric monoclonal antibodies For in vivo use of anti-TRLD in humans, it may be preferable to use "humanized" chimeric monoclonal antibodies. Such antibodies can be produced using genetic constructs derived from hybridoma cells producing the monoclonal antibodies described above. Methods for producing chimeric antibodies are known in the art. See, for review, Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabilly et al., U.S. Patent No.
  • TRLD is expressed in hematopoeitic tissues and other normal human tissues.
  • substantially altered (increased or decreased) levels of TRID gene expression can be detected in immune system tissue or other cells or bodily fluids (e.g., sera and plasma) taken from an individual having such a disorder, relative to a "standard" TRLD gene expression level, that is, the TRLD expression level in immune system tissues or bodily fluids from an individual not having the immune system disorder.
  • the invention provides a diagnostic method useful during diagnosis of an immune system disorder, which involves measuring the expression level of the gene encoding the TRLD protein in immune system tissue or other cells or body fluid from an individual and comparing the measured gene expression level with a standard TRLD gene expression level, whereby an increase or decrease in the gene expression level compared to the standard is indicative of an immune system disorder.
  • certain tissues in mammals with cancer express significantly enhanced levels of the TRLD protein and mRNA encoding the TRLD when compared to a corresponding "standard" level.
  • enhanced levels of the TRID protein can be detected in certain body fluids (e.g., sera and plasma) from mammals with such a cancer when compared to sera from mammals of the same species not having the cancer.
  • the invention provides a diagnostic method useful during diagnosis of an immune system disorder, including cancers which involves measuring the expression level of the gene encoding the TRLD protein in immune system tissue or other cells or body fluid from an individual and comparing the measured gene expression level with a standard TRLD gene expression level, whereby an increase or decrease in the gene expression level compared to the standard is indicative of an immune system disorder.
  • the present invention is useful as a prognostic indicator, whereby patients exhibiting altered (particularly enhanced) gene expression will experience a worse clinical outcome relative to patients expressing the gene at a level nearer the standard level.
  • test the expression level of the gene encoding a TRID protein is intended qualitatively or quantitatively measuring or estimating the level of TRLD or the level of the mRNA encoding TRLD in a first biological sample either directly (e.g., by determining or estimating absolute protein level or mRNA level) or relatively (e.g.. by comparing to the TRLD protein level or mRNA level in a second biological sample).
  • the TRLD protein level or mRNA level in the first biological sample is measured or estimated and compared to a standard TRID protein level or mRNA level, the standard being taken from a second biological sample obtained from an individual not having the disorder or being determined by averaging levels from a population of individuals not having a disorder of the immune system.
  • biological sample any biological sample obtained from an individual, body fluid, cell line, tissue culture, or other source which contains TRLD protein or mRNA.
  • biological samples include body fluids (such as sera, plasma, urine, synovial fluid and spinal fluid) which contain free extracellular domain(s) (or soluable form(s)) of a TRID protein, immune system tissue, and other tissue sources found to express complete or extracellular domain of TRLD. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art. Where the biological sample is to include mRNA, a tissue biopsy is the preferred source.
  • the invention also contemplates the use of a gene of the present invention for diagnosing mutations in the TRLD gene. For example, if a mutation is present in one of the genes of the present invention, conditions would result from a lack of production of the receptor polypeptides of the present invention. Further, mutations which enhance receptor polypeptide activity would lead to diseases associated with an over expression of the receptor polypeptide, e.g., endotoxic shock. Mutations in the genes can be detected by comparing the sequence of the defective gene with that of a normal one. Subsequently one can verify that a mutant gene is associated with a disease condition or the susceptibility to a disease condition. That is, a mutant gene which leads to the overexpression of TRID would be associated with an inability of TRAIL to inhibit tumor growth.
  • immune system disorders which may be diagnosed by the foregoing assays include hypersensitivity, allergy, infectious disease, graft-host disease, immunodeficiency, autoimmune diseases and the like.
  • Nucleic acids used for diagnosis may be obtained from a patient's cells, such as from blood, urine, saliva and tissue biopsy among other tissues.
  • the genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR (Saiki et al., Nature, 324:163-166 (1986)) prior to analysis.
  • RNA or cDNA may also be used for the same purpose.
  • PCR primers complementary to the nucleic acid of the instant invention can be used to identify and analyze mutations in the human genes of the present invention.
  • deletions and insertions can be detected by a change in the size of the amplified product in comparison to the normal genotype.
  • Point mutations can be identified by hybridizing amplified DNA to radiolabeled RNA or alternatively, radiolabeled antisense DNA sequences of the present invention. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase A digestion or by differences in melting temperatures. Such a diagnostic would be particularly useful for prenatal or even neonatal testing. Sequence differences between the reference gene and "mutants" may be revealed by the direct DNA sequencing method.
  • cloned DNA segments may be used as probes to detect specific DNA segments. The sensitivity of this method is greatly enhanced when combined with PCR.
  • a sequencing primary used with double stranded PCR product or a single stranded template molecule generated by a modified PCR product.
  • the sequence determination is performed by conventional procedures with radiolabeled nucleotides or by automatic sequencing procedures with fluorescent tags.
  • Sequence changes at the specific locations may be revealed by nuclease protection assays, such as RNase and S 1 protection or the chemical cleavage method (for example, Cotton et al., PNAS, 85:4397-4401 (1985)).
  • TRLD protein levels in a biological sample can occur using antibody-based techniques.
  • TRLD protein expression in tissues can be studied with classical immunohistological methods (Jalkanen, M., et al, J. Cell. Biol. 101:916-985 (1985); Jalkanen, M., et al, J. Cell . Biol. 105:3081-3096 (1987)).
  • Other antibody-based methods useful for detecting TRLD gene expression include immunoassay s, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA).
  • ELISA enzyme linked immunosorbent assay
  • RIA radioimmunoassay
  • Suitable antibody assay labels include enzyme labels, such as, glucose oxidase, and radioisotopes, such as iodine ( 125 I, I21 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( In), and technetium ( 99m Tc), and fluorescent labels, such as fluorescein and rhodamine, and biotin.
  • enzyme labels such as, glucose oxidase, and radioisotopes, such as iodine ( 125 I, I21 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( In), and technetium ( 99m Tc)
  • fluorescent labels such as fluorescein and rhodamine, and biotin.
  • TRLD proteins can also be detected in vivo by imaging.
  • Antibody labels or markers for in vivo imaging of TRID proteins include those detectable by X-radiography, NMR or ESR.
  • suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not overtly harmful to the subject.
  • suitable markers for NMR and ESR include those with a detectable characteristic spin, such as deuterium, which may be inco ⁇ orated into the antibody by labeling of nutrients for the relevant hybridoma.
  • a TRLD-specific antibody or antibody fragment which has been labeled with an appropriate detectable imaging moiety such as a radioisotope (for example, 131 I, 112 In, 99m Tc), a radio-opaque substance, or a material detectable by nuclear magnetic resonance, is introduced (for example, parenterally, subcutaneously or intraperitoneally ) into the mammal to be examined for immune system disorder.
  • a radioisotope for example, 131 I, 112 In, 99m Tc
  • a radio-opaque substance for example, parenterally, subcutaneously or intraperitoneally
  • the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of 99m Tc.
  • the labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain TRID protein.
  • In vivo tumor imaging is described in S.W. Burchiel et al., "Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments" (Chapter 13 in C Tumor Imaging: The Radiochemical Detection of Cancer, S.W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982)).
  • Tumor Necrosis Factor (TNF) family ligands are known to be among the most pleiotropic cytokines, inducing a large number of cellular responses, including cytotoxicity, anti-viral activity, immunoregulatory activities, and the transcriptional regulation of several genes (Goeddel, D.V. et al, "Tumor Necrosis Factors: Gene Structure and Biological Activities," Symp. Quant. Biol. 51:591-609 (1986), Cold Spring Harbor; Beutler, B., and Cerami, A., Annu. Rev. Biochem. 57:505-518 (1988); Old, L.J., Sci. Am. 255:59-75 (1988); Fiers, W., FEBS Lett.
  • TNF Tumor Necrosis Factor
  • the TNF-family ligands induce such various cellular responses by binding to TNF-family receptors.
  • Cells which express a TRLD polypeptide and have a potent cellular response to TNFR ligands include lymphocytes, endothelial cells, keratinocytes, and prostate tissue.
  • a cellular response to a TNF-family ligand is intended any genotypic, phenotypic, and/or mo ⁇ hologic change to a cell, cell line, tissue, tissue culture or patient that is induced by a TNF-family ligand.
  • such cellular responses include not only normal physiological responses to TNF-family ligands, but also diseases associated with increased apoptosis or the inhibition of apoptosis.
  • Diseases associated with increased cell survival, or the inhibition of apoptosis include cancers (such as follicular lymphomas, carcinomas with p53 mutations, and hormone- dependent tumors, such as breast cancer, prostate cancer, Kaposi's sarcoma and ovarian cancer); autoimmune disorders (such as systemic lupus erythematosus and immune-related glomerulonephritis rheumatoid arthritis) and viral infections (such as he ⁇ es viruses, pox viruses and adenoviruses), information graft v. host disease, acute graft rejection, and chronic graft rejection.
  • Diseases associated with increased apoptosis include ALDS; neurodegeneradve disorders (such as Alzheimer's disease.
  • Parkinson's disease Amyotrophic lateral sclerosis, Retinitis pigmentosa, Cerebellar degeneration
  • myelodysplastic syndromes such as aplastic anemia
  • ischemic injury such as that caused by myocardial infarction, stroke and reperfusion injury
  • toxin-induced liver disease such as that caused by alcohol
  • septic shock cachexia and anorexia.
  • the present invention is directed to a method for enhancing apoptosis induced by a TNF-family ligand, which involves administering to a cell which expresses a TNFR polypeptide an effective amount of an antagonist of the TR D polypeptide, capable of inhibiting TRLD expression or its ligand binding ability (e.g., to TRAIL).
  • an antagonist of the TR D polypeptide capable of inhibiting TRLD expression or its ligand binding ability (e.g., to TRAIL).
  • TNFR mediated signaling is increased to treat a disease wherein decreased apoptosis is exhibited.
  • Antagonist can include monoclonal antibodies directed against the TRLD polypeptide.
  • antagonist is intended naturally occurring and synthetic compounds capable of enhancing or potentiating apoptosis.
  • agonist is intended naturally occurring and synthetic compounds capable of inhibiting apoptosis. Whether any candidate "antagonist” or “agonist” of the present invention can enhance or inhibit apoptosis can be determined using art-known TNF-family ligand/receptor cellular response assays, including those described in more detail below.
  • One such screening procedure involves the use of melanophores which are transfected to co-express a TNFR receptor which binds a TRAIL such as DR4 or DR5, described elsewhere herein, and the TRID receptor of the present invention.
  • a screening technique is described in PCT WO 92/01810, published February 6, 1992.
  • Such an assay may be employed, for example, for screening for a compound which inhibits (or enhances) the activity of the receptor polypeptide of the present invention by contacting the melanophore cells which encode the receptors with both a TNF-family ligand and the candidate antagonist (or agonist). Inhibition or enhancement of the signal generated by the ligand indicates that the compound is an antagonist or agonist of TRLD activity.
  • the TRID polypeptide and its agonists inhibit activation of the TNFR receptor, e.g. , TRAIL, receptor, whereas antagonists will increase activation.
  • TRAIL receptor and TRLD for example, transfected CHO cells
  • compounds may be contacted with a cell which expresses a TRAIL receptor polypeptide and TRID of the present invention and a second messenger response, e.g., signal transduction or pH changes, may be measured to determine whether the potential compound activates or inhibits the TRAIL receptor.
  • a second messenger response e.g., signal transduction or pH changes
  • Another such screening technique involves introducing RNA encoding the receptors into Xenopus oocytes to transiently express TRID and a TRAIL receptor.
  • the receptor oocytes may then be contacted with the receptor ligand and a compound to be screened, followed by detection of inhibition or activation of a calcium signal in the case of screening for compounds which are thought to inhibit activation of the receptor.
  • Another screening technique involves expressing in cells a construct wherein the
  • TRAIL receptor is linked to a phospholipase C or D.
  • Such cells include endothelial cells, smooth muscle cells, embryonic kidney cells, etc.
  • the screening may be accomplished as hereinabove described by detecting activation of the receptor or inhibition of activation of the receptor from the phospholipase signal in the presence of TRID either co-expressed or added in soluble form along with the candidate compound.
  • Another method involves screening for compounds which inhibit activation of a TRAIT , receptor polypeptide in the presence of the TRID polypeptide of the present invention, either co-expressed or in soluble form.
  • Agonists of the present invention are identified by
  • TRAIL can be labeled, e.g., by radioactivity.
  • the amount of labeled TRAIL bound to the receptors is measured, e.g., by measuring radioactivity of the receptors. If the compound binds to the TRLD receptor as determined by an increase of labeled TRAIL which binds to the TRAIL receptor, the compound is a TRID antagonist.
  • a screening method for determining whether a candidate TRID antagonist or agonist is capable of enhancing or inhibiting a cellular response to a TNF-family ligand (e.g., apoptosis induced by TRAIL).
  • a TNF-family ligand e.g., apoptosis induced by TRAIL
  • the method involves contacting cells which express a TNFR polypeptide with a candidate compound, TRID, and a TNF-family ligand, assaying a cellular response, and comparing the cellular response to a standard cellular response, the standard being assayed when contact is made with the ligand in the presence of TRLD but in absence of the candidate compound, whereby an increased cellular response over the standard indicates that the candidate compound is an antagonist and a decreased cellular response compared to the standard indicates that the candidate compound is an agonist.
  • assaying a cellular response is intended qualitatively or quantitatively measuring a cellular response to a candidate compound and or a TNF-family ligand (e.g...
  • a cell expressing the TNFR polypeptide can be contacted with either an endogenous or exogenously administered TNF-family ligand.
  • Antagonist include naturally occurring and synthetic compounds such as, for example, TNF family ligand peptide fragments, transforming growth factor, neurotransmitters (such as glutamate, dopamine, N-mefhyl-D-aspartate), tumor suppressors (p53), cytolytic T cells and antimetabolites.
  • Preferred agonist include chemotherapeutic drugs such as, for example, cisplatin, doxorubicin, bleomycin, cytosine arabinoside, nitrogen mustard, methotrexate and vincristine. Others include ethanol and - amyloid peptide. (Science 267:1457-1458 (1995)). Further preferred antagonist includes polyclonal and monoclonal antibodies raised against the TRID polypeptide, or a fragment thereof.
  • Agonists according to the present invention include naturally occurring and synthetic compounds such as, for example, the CD40 ligand, neutral amino acids, zinc, estrogen, androgens, viral genes (such as Adenovirus EIB, Baculovirus p35 and IAP, Cowpox virus crmA, Epstein-Barr virus BHRF1, LMP-1, African swine fever virus LMW5-HL, and He ⁇ esvirus yl 34.5), calpain inhibitors, cysteine protease inhibitors, and tumor promoters (such as PMA. Phenobarbital, and -Hexachlorocyclohexane).
  • Other Agonists include polyclonal and monoclonal antagonist antibodies raised against TRAIL polypeptides or a fragment thereof.
  • Antisense molecules can be used to control gene expression through antisense DNA or RNA or through triple-helix formation. Antisense techniques are discussed, for example, in Okano, J. Neurochem. 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988). Triple helix formation is discussed in, for instance Lee et al, Nucleic Acids Research 10-1573 (1979); Cooney et al, Science 241:456 (1988); and Dervan et al, Science 257:1360 (1991). The methods are based on binding of a polynucleotide to a complementary DNA or RNA.
  • the 5' coding portion of a polynucleotide that encodes the mature polypeptide of the present invention may be used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length.
  • a DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription thereby preventing transcription and the production of the receptor.
  • the antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into receptor polypeptide.
  • the oligonucleotides described above can also be delivered to cells such that the antisense RNA or DNA may be expressed in vivo to inhibit production of the receptor.
  • Further agonist according to the present invention include soluble forms of TRID, i.e.,
  • TRID fragments that include the ligand binding domain from the extracellular region of the full- length receptor.
  • Such soluble forms of the receptor which may be naturally occurring or synthetic, antagonize TNFR mediated signaling by competing with the cell surface TNFR for binding to TNF-family ligands.
  • soluble forms of the TRLD receptor that include the ligand binding domain are novel cytokines capable of inhibiting apoptosis induced by TNF- family ligands.
  • Other such cytokines are known in the art and include Fas B (a soluble form of the mouse Fas receptor) that acts physiologically to limit apoptosis induced by Fas ligand (Hughes, D.P. and Crispe, I.N., J. Exp. Med. 752: 1395-1401 (1995)).
  • polyclonal and monoclonal antibody agonist or antagonist according to the present invention can be raised according to the methods disclosed in Tartaglia, L.A., and Goeddel, D.V., J. Biol. Chem. 267(7 ):4304-4301 (1992): Tartaglia, L.A. et al, Cell 75:213- 216 (1993), and PCT Application WO 94/09137.
  • antibody or “monoclonal antibody” (mAb) as used herein is meant to include intact molecules as well as fragments thereof (such as, for example, Fab and F(ab') 2 fragments) which are capable of binding an antigen.
  • Fab and F fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding of an intact antibody (Wahl et al, J. Nucl. Med. 24:316-325 (1983)).
  • ⁇ 3Y Antibodies according to the present invention may be prepared by any of a variety of methods described above, and known in the art
  • Proteins and other compounds which bind the extracellular domains are also candidate agonist and antagonist according to the present invention.
  • binding compounds can be "captured” using the yeast two-hybrid system (Fields and Song, Nature 340:245-246 (1989)).
  • yeast two-hybrid system Fields and Song, Nature 340:245-246 (1989)
  • a modified version of the yeast two-hybrid system has been described by Roger Brent and his colleagues (Gyuris, J. et al, Cell 75:791-803 (1993); Zervos, A.S. et al, Cell 72:223-232 (1993)).
  • TNF-family ligand is intended naturally occurring, recombinant, and synthetic ligands that are capable of binding to a member of the TNF receptor family and inducing the ligand/receptor signaling pathway.
  • TNF ligand family include, but are not limited to, TRID ligands, TRAIL, TNF- ⁇ , lymphotoxin- ⁇ (LT- ⁇ , also known as TNF- ⁇ ), LT- ⁇ , FasL, CD40, CD27, CD30, 4-lBB, OX40 and nerve growth factor (NGF).
  • TNF-family ligand include, but are not limited to, TRID ligands, TRAIL, TNF- ⁇ , lymphotoxin- ⁇ (LT- ⁇ , also known as TNF- ⁇ ), LT- ⁇ , FasL, CD40, CD27, CD30, 4-lBB, OX40 and nerve growth factor (NGF).
  • HFV-induced apoptotic cell death has been demonstrated not only in vitro but also, more importantly, in infected individuals (Ameisen, J.C., AIDS 5:1197- 1213 (1994) ; Finkel, T.H., and Banda, N.K., Curr. Opin. Immunol. 6:605-615(1995); Muro-Cacho, CA. et al, J. Immunol. 154:5555-5566 (1995)). Furthermore, apoptosis and CD4 + T-lymphocyte depletion is tightly correlated in different animal models of AIDS (Brunner, T., et al, Nature 373:441-444 (1995); Gougeon, M.L..
  • a method for treating HIV + individuals involves administering soluble TRID (e.2., the extracellular domain) or an agonist of the present invention to reduce
  • the immune system of the recipient animal In rejection of an allograft, the immune system of the recipient animal has not previously been primed to respond because the immune system for the most part is only primed by environmental antigens. Tissues from other members of the same species have not been presented in the same way that, for example, viruses and bacteria have been presented.
  • immunosuppressive regimens are designed to prevent the immune system from reaching the effector stage.
  • the immune profile of xenograft rejection may resemble disease recurrence more that allograft rejection.
  • the immune system In the case of disease recurrence, the immune system has already been activated, as evidenced by destruction of the native islet cells. Therefore, in disease recurrence the immune system is already at the effector stage.
  • Antagonist of the present invention are able to suppress the immune response to both allografts and xenografts because lymphocytes activated and differentiated into effector cells will express TNFR polypeptides, and thereby are susceptible to compounds which enhance TNFR activity.
  • the present invention further provides a method for creating immune privileged tissues.
  • Agonist of the invention can further be used in the treatment of Inflammatory Bowel-Disease.
  • TRID polypeptide composition will be formulated and dosed in a fashion consistent with good medical practice, taking into account the clinical condition of the individual patient (especially the side effects of treatment with TRLD polypeptide alone), the site of delivery of the TRID polypeptide composition, the method of administration, the scheduling of administration, and other factors known to practitioners.
  • the "effective amount" of TRLD polypeptide for pu ⁇ oses herein is thus determined by such considerations.
  • the total pharmaceutically effective amount of TRLD polypeptide administered parenterally per dose will be in the range of about 1 ⁇ g/kg/day to 10 mg/kg/day of patient body weight, although, as noted above, this will be subject to therapeutic discretion. More preferably, this dose is at least 0.01 mg/kg/day, and most preferably for humans between about 0.01 and 1 mg kg/day for the hormone.
  • the TRLD polypeptide is typically administered at a dose rate of about 1 ⁇ g/kg/hour to about 50 ⁇ g/kg/hour, either by 1-4 injections per day or by continuous subcutaneous infusions, for example, using a mini-pump. An intravenous bag solution may also be employed. The length of treatment needed to observe changes and the interval following treatment for responses to occur appears to vary depending on the desired effect.
  • compositions containing the TRID of the invention may be administered orally, rectally, parenterally, intracistemally, intravaginally. intraperitoneally, topically (as by powders, ointments, drops or transdermal patch), bucally, or as an oral or nasal spray.
  • pharmaceuticallyv acceptable carrier is meant a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • parenteral refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.
  • the TRLD polypeptide is also suitably administered by sustained-release systems.
  • sustained-release compositions include semi-permeable polymer matrices in the form of shaped articles, e.g., films, or mirocapsules.
  • Sustained-release matrices include polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman, U. et al., Biopolymers 22:541-556 (1983)), poly (2- hydroxyethyl methacrylate) (R. Langer et al., J. Biomed. Mater. Res.
  • Sustained-release TRLD polypeptide compositions also include liposomally entrapped TRLD polypeptides. Liposomes containing TRID polypeptides are prepared by methods known per se: DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. (USA) 82:3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci.
  • the liposomes are of the small (about 200-800 Angstroms) unilamellar type in which the lipid content is greater than about 30 mol. percent cholesterol, the selected proportion being adjusted for the optimal TNFR polypeptide therapy.
  • the TRLD polypeptide is formulated generally by mixing it at the desired degree of purity, in a unit dosage injectable form (solution, suspension, or emulsion), with a pharmaceutically acceptable carrier, i.e., one that is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation.
  • a pharmaceutically acceptable carrier i.e., one that is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation.
  • the formulation preferably does not include oxidizing agents and other compounds that are known to be deleterious to polypeptides.
  • the formulations are prepared by contacting the TRID polypeptide uniformly and intimately with liquid carriers or finely divided solid carriers or both. Then, if necessary, the product is shaped into the desired formulation.
  • the carrier is a parenteral carrier, more preferably a solution that is isotonic with the blood of the recipient. Examples of such carrier vehicles include water, saline, Ringer's solution, and dextrose solution. Non-aqueous vehicles such as fixed oils and ethyl oleate are also useful herein, as well as liposomes.
  • the carrier suitably contains minor amounts of additives such as substances that enhance isotonicity and chemical stability.
  • additives such as substances that enhance isotonicity and chemical stability.
  • Such materials are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, succinate. acetic acid, and other organic acids or their salts: antioxidants such as ascorbic acid; low molecular weight (less than about ten residues) polypeptides, e.g., polyarginine or tripeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid, aspartic acid, or arginine: monosaccharides, disaccharides. and other carbohydrates including cellulose
  • 77 or its derivatives, glucose, manose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions such as sodium; and/or nonionic surfactants such as polysorbates, poloxamers, or PEG.
  • chelating agents such as EDTA
  • sugar alcohols such as mannitol or sorbitol
  • counterions such as sodium
  • nonionic surfactants such as polysorbates, poloxamers, or PEG.
  • TRLD polypeptide is typically formulated in such vehicles at a concentration of about 0.1 mg/ml to 100 mg/ml, preferably 1-10 mg/ml, at a pH of about 3 to 8. It will be understood that the use of certain of the foregoing excipients, carriers, or stabilizers will result in the formation of TRLD polypeptide salts.
  • TRID polypeptides to be used for therapeutic administration must be sterile. Sterility is readily accomplished by filtration through sterile filtration membranes (e.g., 0.2 micron membranes). Therapeutic TRID polypeptide compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
  • a sterile access port for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
  • TRLD polypeptides ordinarily will be stored in unit or multi-dose containers, for example, sealed ampoules or vials, as an aqueous solution or as a lyophilized formulation for reconstitution.
  • a lyophilized formulation 10-ml vials are filled with 5 ml of sterile-filtered 1% (w/v) aqueous TRID polypeptide solution, and the resulting mixture is lyophilized.
  • the infusion solution is prepared by reconstituting the lyophilized TRLD polypeptide using bacteriostatic Water-for-Injection.
  • the invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
  • a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
  • Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • the polypeptides of the present invention may be employed in conjunction with other therapeutic compounds.
  • the nucleic acid molecules of the present invention are also valuable for chromosome identification.
  • the sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome.
  • Few chromosome marking reagents based on actual sequence data (repeat polymo ⁇ hisms) are presently available for marking chromosomal location.
  • the mapping of DNAs to chromosomes according to the present invention is an important first step in correlating those sequences with genes associated with disease.
  • the cDNAs herein disclosed are used to clone genomic DNA of a TRID protein gene. This can be accomplished using a variety of well known techniques and libraries, which generally are available commercially. The genomic DNA then is used for in situ chromosome mapping using well known techniques for this purpose. ⁇ ⁇ In addition, in some cases, sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the cDNA. Computer analysis of the 3' untranslated region of the gene is used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes.
  • Fluorescence in situ hybridization of a cDNA clone to a metaphase chromosomal spread can be used to provide a precise chromosomal location in one step.
  • This technique can be used with probes from the cDNA as short as 50 or 60 bp.
  • Verma et al Human Chromosomes: A Manual Of Basic Techniques, Pergamon Press, New York (1988).
  • the bacterial expression vector pQE9 (pDIO) is used for bacterial expression in this example.
  • pQE9 encodes ampicillin antibiotic resistance ("Ampr") and contains a bacterial origin of replication ("ori"), an IPTG inducible promoter, a ribosome binding site (“RBS”), six codons encoding histidine residues that allow affinity purification using nickel-nitrilo-tri-acetic acid (“Ni-NTA”) affinity resin sold by QIAGEN, Inc., supra, and suitable single restriction enzyme cleavage sites.
  • These elements are arranged such that an inserted DNA fragment encoding a polypeptide expresses that polypeptide with the six His residues (i.e., a "6 X His tag”) covalently linked to the amino terminus of that polypeptide.
  • the DNA sequence encoding the desired portion of the TRLD protein comprising the extracellular form of the TRID amino acid sequence is amplified from the deposited cDNA clone using PCR oligonucleotide primers which anneal to sequence encoding the amino terminal sequences of the desired portion of the TRID protein and to carboxy terminal sequences of the desired portion of the extracellular form of the TRID protein in the deposited cDNA. Additional nucleotides containing restriction sites to facilitate cloning in the pQE9 vector are added to the 5' and 3' primer sequences, respectively.
  • the 5' primer has the sequence 5' CGCGGATCCACCACTGCCCGGCAGGAG 3TSEO ED NO: 19) containing the underlined BamHI restriction site followed by 18 nucleotides of the amino terminal coding sequence of the extracellular TRID sequence in SEQ ID NO:2.
  • the point in the protein coding sequence where the 5' primer begins and where the 3' primer ends may be varied to amplify a DNA segment encoding any desired portion of the complete TRLD protein shorter or longer than the extracellular form of the protein.
  • the 3' primer has the sequence
  • the amplified TRID DNA fragment and the vector pQE9 are digested with BamHI and Xbal and the digested DNAs are then ligated together. Insertion of the TRLD DNA into the restricted pQE9 vector places the TRLD protein coding region downstream from the IPTG- inducible promoter and in-frame with an initiating AUG and the six histidine codons.
  • E. coli strain M15/rep4 containing multiple copies of the plasmid pREP4, which expresses the lac repressor and confers kanamycin resistance ("Kanr"), is used in carrying out the illustrative example described herein.
  • This strain which is only one of many that are suitable for expressing TRLD protein, is available commercially from QIAGEN, Inc., supra. Transformants are identified by their ability to grow on LB plates in the presence of ampicillin and kanamycin. Plasmid DNA is isolated from resistant colonies and the identity of the cloned DNA confirmed by restriction analysis, PCR and DNA sequencing.
  • Clones containing the desired constructs are grown overnight ("O/N") in liquid culture in LB media supplemented with both ampicillin (100 ⁇ g/ml) and kanamycin (25 ⁇ g/ml).
  • the O/N culture is used to inoculate a large culture, at a dilution of approximately 1:25 to 1 :250.
  • the cells are grown to an optical density at 600 nm ("OD600") of between 0.4 and 0.6.
  • Isopropyl- ⁇ -D-thiogalactopyranoside (“LPTG”) is then added to a final concentration of 1 mM to induce transcription from the lac repressor sensitive promoter, by inactivating the lacl repressor.
  • Cells subsequently are incubated further for 3 to 4 hours. Cells then are harvested by centrifugation.
  • Ni-NTA nickel-nitrilo-tri-acetic acid
  • the column is first washed with 10 volumes of 6 M guanidine-HCl, pH 8, then washed with 10 volumes of 6 M guanidine-HCl pH 6, and finally the TRLD is eluted with 6 M guanidine-HCl, pH 5.
  • the purified protein is then renatured by dialyzing it against phosphate-buffered saline (PBS) or 50 mM Na-acetate, pH 6 buffer plus 200 mM NaCl.
  • PBS phosphate-buffered saline
  • the protein can be successfully refolded while immobilized on the Ni-NTA column.
  • the recommended conditions are as follows: renature using a linear 6M-1M urea gradient in 500 mM NaCl, 20% glycerol. 20 mM Tris/HCl pH 7.4, containing protease inhibitors.
  • the renaturation should be performed over a period of 1.5 hours or more.
  • the proteins can be eluted by the addition of 250 mM immidazole. Immidazole is removed by a final dialyzing step against PBS or 50 mM sodium acetate pH 6 buffer plus 200 mM NaCl.
  • the purified protein is stored at 4° C or frozen at -80° C
  • the plasmid shuttle vector pA2 is used to insert the cloned DNA encoding complete protein, including its naturally associated secretory signal (leader) sequence, into a baculovirus to express the mature TRID protein, using standard methods as described in Summers et al. , A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures, Texas Agricultural Experimental Station Bulletin No. 1555 (1987).
  • This expression vector contains the strong polyhedrin promoter of the Autographa califomica nuclear polyhedrosis virus (AcMNPV) followed by convenient restriction sites such as BamHI, Xba I and Asp718.
  • the polyadenylation site of the simian virus 40 (“SV40") is used for efficient polyadenylation.
  • the plasmid contains the beta-galactosidase gene from E. coli under control of a weak Drosophila promoter in the same orientation, followed by the polyadenylation signal of the polyhedrin gene.
  • the inserted genes are flanked on both sides by viral sequences for cell-mediated homologous recombination with wild-type viral DNA to generate a viable virus that express the cloned polynucleotide.
  • Many other baculovirus vectors could be used in place of the vector above, such as pAc373, pVL941 and pAcIMl.
  • the cDNA sequence encoding the full length TRLD protein in a deposited clone, including the AUG initiation codon and the naturally associated leader sequence shown in SEQ LD NO:2 is amplified using PCR oligonucleotide primers corresponding to the 5' and 3' sequences of the gene.
  • the 5' primer for TRLD has the sequence 5' CGCTCTAGACCGCCATCATGGCCCGGATCCCCAAG 3' (SEQ LD NO:21) containing the underlined Xbal restriction enzyme site.
  • the described primers encode an efficient signal for initiation of translation in eukaryotic cells, as described by Kozak, M., J. Mol. Biol. 196:941-950 (1987).
  • the 3' primer for TRLD has the sequence
  • the amplified fragment is isolated from a 1% agarose gel using a commercially available kit ("Geneclean,” BIO 101 Inc., La Jolla, Ca.).
  • the fragment then is digested with the appropriate restriction enzyme for each of the primers used, as specified above, and again is purified on a 1 % agarose gel.
  • the plasmid is digested with the same restriction enzymes and optionally, can be dephosphorylated using calf intestinal phosphatase. using routine procedures known in the art.
  • the DNA is then isolated from a 1% agarose gel using a commercially available kit ("Geneclean" BIO 101 Inc., La Jolla, Ca.).
  • the fragment and dephosphorylated plasmid are ligated together with T4 DNA ligase.
  • E. coli HB101 or other suitable E. coli hosts such as XL-1 Blue (Statagene Cloning Systems, La Jolla, CA) cells are transformed with the ligation mixture and spread on culture plates.
  • Bacteria are identified that contain the plasmid with the human TNF receptor gene by digesting DNA from individual colonies using the enzymes used immediately above and then analyzing the digestion product by gel electrophoresis. The sequence of the cloned fragment is confirmed by DNA sequencing. This plasmid is designated herein pA2-TRTD.
  • plasmid pA2-TRLD Five ⁇ g of the plasmid pA2-TRLD is co-transfected with 1.0 ⁇ g of a commercially available linearized baculovirus DNA ("BaculoGoldTM baculovirus DNA", Pharmingen. San Diego, CA), using the lipofection method described by Feigner et al., Proc. Natl. Acad. Sci. USA 84: 7413-7417 (1987).
  • BaculoGoldTM virus DNA and 5 ⁇ g of the plasmid pA2-TNFR are mixed in a sterile well of a microtiter plate containing 50 ⁇ l of serum-free Grace's medium (Life Technologies Inc., Gaithersburg. MD).
  • plaque assay of this type can also be found in the user's guide for insect cell culture and baculovirology distributed by Life Technologies Inc., Gaithersburg, page 9-10). After appropriate incubation, blue stained plaques are picked with the tip of a micropipettor (e.g., Eppendorf). The agar containing the recombinant viruses is then resuspended in a microcentrifuge tube containing 200 ⁇ l of Grace's
  • V-TRID The recombinant virus
  • Sf9 cells are grown in Grace's medium supplemented with 10% heat-inactivated FBS.
  • the cells are infected with the recombinant o baculovirus V-TRID at a multiplicity of infection ("MOI") of about 2.
  • MOI multiplicity of infection
  • the medium is removed and is replaced with SF900 II medium minus methionine and cysteine (available from Life Technologies Inc.. Rockville, MD).
  • 5 ⁇ Ci of 33 S -methionine and 5 ⁇ Ci 35 S-cysteine available from Amersham
  • the cells are further incubated for 16 hours and then are harvested by centrifugation.
  • the 5 proteins in the supernatant as well as the intracellular proteins are analyzed by SDS-PAGE followed by autoradiography (if radiolabeled).
  • Microsequencing of the amino acid sequence of the amino terminus of purified protein may be used to determine the amino terminal sequence of the mature protein and thus the cleavage point and length of the secretory signal peptide.
  • a typical mammalian expression vector contains the promoter element, which mediates the initiation of transcription of mRNA, the protein coding sequence, and signals required for the termination of transcription and polyadenylation of the transcript. Additional elements include enhancers. Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing. Highly efficient transcription can be achieved with the early and late
  • V3 promoters from SV40 the long terminal repeats (LTRs) from Retroviruses, e.g., RSV, HTLVI, HI VI and the early promoter of the cytomegalovirus (CMV).
  • LTRs long terminal repeats
  • CMV cytomegalovirus
  • cellular elements can also be used (e.g., the human actin promoter).
  • Suitable expression vectors for use in practicing the present invention include, for example, vectors such as pSVL and pMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146) and pBC12MI (ATCC 67109).
  • Mammalian host cells that could be used include: human Hela 293, H9 and Jurkat cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7 and CVI, quail QC1-3 cells, mouse L cells and Chinese hamster ovary (CHO) cells.
  • the gene can be expressed in stable cell lines that contain the gene integrated into a chromosome.
  • a selectable marker such as dhfr, gpt, neomycin, hygromycin allows the identification and isolation of the transfected cells.
  • the transfected gene can also be amplified to express large amounts of the encoded protein.
  • the dihydrofolate reductase (DHFR) marker is useful to develop cell lines that carry several hundred or even several thousand copies of the gene of interest.
  • Another useful selection marker is the enzyme glutamine synthase (GS) (Mu ⁇ hy et al., Biochem J. 227:211- 279 (1991): Bebbington et al., Bio/Technology 10:169- 15 (1992)). Using these markers, the mammalian cells are grown in selective medium and the cells with the highest resistance are selected. These cell lines contain the amplified gene(s) integrated into a chromosome. Chinese hamster ovary (CHO) and NSO cells are often used for the production of proteins.
  • the expression vectors pCl and pC4 contain the strong promoter (LTR) of the Rous
  • the expression plasmid, pTRID-HA is made by cloning a cDNA encoding TRLD into the expression vector pcDNAI/Amp or pcDNAIII (which can be obtained from Invitrogen, Inc.).
  • the expression vector pcDNAI/Amp contains: (1) an E. coli origin of replication effective for propagation in E. coli and other prokaryotic cells; (2) an ampicillin resistance gene for selection of plasmid-containing prokaryotic cells; (3) an SV40 origin of replication for propagation in eukaryotic cells; (4) a CMV promoter, a polylinker.
  • a DNA fragment encoding the TRLD is cloned into the polylinker region of the vector so that recombinant protein expression is directed by the CMV promoter.
  • the plasmid construction strategy is as follows.
  • the TRLD cDNA of the deposited clone is amplified using primers that contain convenient restriction sites, much as described above for construction of vectors for expression of a TNF receptor in E. coli. Suitable primers include the following, which are used in this example.
  • the 5' primer for TNFR-5 containing the underlined EcoRI site, has the following sequence: 5 ' CGCGAATTCCGCCATCATGGCCCGGATCCCCAAG 3' (SEQ ID NO:23).
  • the 3' primer containing the underlined Xbal site, has the following sequence: 5' GCGTCTAGAGTAATGAGAAGAGGCAGG 3' (SEQ ID NO:24).
  • COS cells are transfected with an expression vector, as described above, using DEAE-DEXTRAN, as described, for instance, in Sambrook et al, Molecular Cloning: a Laboratory Manual, Cold Spring Laboratory Press, Cold Spring Harbor, New York (1989). Cells are incubated under conditions for expression of TRLD by the vector.
  • Plasmid pC4 is used for the expression of TRLD polypeptides.
  • Plasmid pC4 is a derivative of the plasmid pSV2-dhfr (ATCC Accession No. 37146).
  • the plasmid contains the mouse DHFR gene under control of the SV40 early promoter.
  • Chinese hamster ovary- or other cells lacking dihydrofolate activity that are transfected with these plasmids can be selected by growing the cells in a selective medium (alpha minus MEM, Life Technologies) supplemented with the chemotherapeutic agent methotrexate.
  • MTX methotrexate
  • Plasmid pC4 contains for expressing the gene of interest the strong promoter of the long terminal repeat (LTR) of the Rous Sarcoma Virus (Cullen et al., Molecular and Cellular Biology 5:438-447 (1985) plus a fragment isolated from the enhancer of the immediate early gene of human cytomegalovirus (CMV) (Boshart et al., Cell 47:521-530 (1985)).
  • LTR long terminal repeat
  • CMV cytomegalovirus
  • Downstream of the promoter are the following single restriction enzyme cleavage sites that allow the integration of the genes: BamHI, Xba I, and Asp718. Behind these cloning sites the plasmid contains the 3' intron and polyadenylation site of the rat preproinsulin gene.
  • Other high efficiency promoters can also be used for the expression, e.g., the human ⁇ -actin promoter, the SV40 early or late promoters or the long terminal repeats from other retroviruses, e.g., HTV and HTLVI.
  • the plasmid pC4 is digested with the restriction enzymes appropriate for the specific primers used to amplify TRLD as outlined below and then dephosphorylated using calf intestinal phosphates by procedures known in the art.
  • the vector is then isolated from a 1% agarose gel.
  • the DNA sequence encoding the TRLD polypeptide is amplified using PCR oligonucleotide primers corresponding to the 5' and 3' sequences of the desired portion of the gene.
  • the 5' primer for TRID containing the underlined Xbal site has the following sequence: 5' CGCICTAGACCGCCATCATGGCCCGGATCCCCAAG 3' (SEQ ID NO:25).
  • the 3' primer for TRID containing the underlined Xbal site, has the following sequence: 5' GCGTCTAGACTAGTAATGAGAAGAGGCAGG 3' (SEQ ID NO:26).
  • the amplified fragment is digested with the endonucleases which will cut at the engineered restriction site(s) and then purified again on a 1% agarose gel.
  • the isolated fragment and the dephosphorylated vector are then ligated with T4 DNA ligase.
  • E. coli HB 101 or XL-1 Blue cells are then transformed and bacteria are identified that contain the fragment inserted into plasmid pC4 using, for instance, restriction enzyme analysis.
  • Northern blot analysis was carried out to examineTRID gene expression in human tissues, using methods described by, among others, Sambrook et al, cited above.
  • a cDNA probe containing the entire nucleotide sequence of the TRLD protein (SEQ ID NO: l) was labeled with 32 P using the red/primeTM DNA labeling system (Amersham Life Science), according to manufacturer's instructions. After labeling, the probe was purified using a CHROMA SPIN- 100TM column (Clontech Laboratories, Inc.), according to manufacturer's protocol number PT 1200-1. The purified labeled probe was then used to examine various human tissues for TRID mRNA.
  • TRID Tumor necrosis virus
  • HL60 promyelocytic leukemia
  • Hela cell S3, K562 chronic myelogeneous leukemia
  • MOLT4 lymphoblast leukemia
  • Raji Bovine lymphoma
  • SW480 colonrectal adenocarcinoma
  • A549 lung carcinoma
  • G361 melanoma
  • TRALL/Apo2L is a cytotoxic ligand that belongs to the tumor necrosis factor (TNF) ligand family and induces rapid cell death of many transformed cell lines, but not normal tissues, despite its death domain containing receptor, DR4, being expressed on both cell types.
  • TNF tumor necrosis factor
  • This example identifies an antagonist decoy receptor, designated "TRAIT . Receptor Without Intracellular Domain” or "TRLD”. that also binds TRAIL and may in part explain the resistant phenotype of normal tissues. That is, TRID, an antagonistic receptor, binds and sequesters TRAIL, but is incapable of transducing an intracellular signal.
  • TRID extracellular ligand binding cysteine-rich domains of TRID and DR4
  • the present inventors theorized that TRID would also bind TRAIL.
  • the soluble extracellular ligand binding domain of TRLD was expressed as a fusion to the Fc portion of human immunoglobulin (IgG).
  • IgG human immunoglobulin
  • TRID-Fc specifically bound TRAIL, but not the related cytotoxic ligand TNF ⁇ .
  • TNFR1 and the corresponding ligands were prepared and binding assays performed as described in Pan et al, Science 276: 1 1 ( 1997).
  • the respective Fc-fusions were precipitated with protein G-Sepharose and co-precipitated soluble ligands were detected by immunoblotting with anti-Rag (Babco) or anti-myc-HRP (BMB).
  • the bottom panel of Fig. 5A shows the input Fc-fusions present in the binding assays.
  • TRID-Fc blocked the ability of TRAIL to induce apoptosis (Fig. 5B).
  • TNFRl-Fc completely abolished TNF ⁇ killing (Fig 5C).
  • MCF7 cells were treated with TNF ⁇ (40 ng/ml; Genentech, Inc.) in the presence of equal amounts of Fc-fusions or Fc alone. Nuclei were stained and examined 1 1-15 hours later.
  • cells expressing TRID were protected from TRAIL-induced apoptosis as were cells expressing the virally encoded caspase inhibitor CrmA.
  • TRLD TRALL-sensitive cells
  • Overexpression of TRID by itself did not induce apoptosis.
  • cells expressing TRLD were as protected from TRAIL-induced apoptosis as were cells expressing the virally encoded caspase inhibitor CrmA (Fig. 6).
  • ADDRESSEE HUMAN GENOME SCIENCES, INC.
  • MOLECULE TYPE DNA (genomic)
  • Trp Asp Asp lie Gin Cys Val Glu Glu Phe Gly Ala Asn Ala Thr Val 115 120 125
  • GCT GCT GAA GAG ACA ATG ACC ACC AGC CCG
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • CAANGTTTCC AANATGNACT TTTTNGTTCC CTGTTANATT TTTTAATTAG TTNAANTTAA 480 ATTTNTNAAC CTTNCCNGGG NAAATT 506
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • SEQUENCE DESCRIPTION SEQ ID NO: 25: CGCTCTAGAC CGCCATCATG GCCCGGATCC CCAAG 35
  • MOLECULE TYPE DNA (genomic)

Abstract

La présente invention concerne un nouveau gène humain qui code un polypeptide membre de la famille des récepteurs du TNF et dont on sait aujourd'hui qu'il lie à la molécule TRAIL. Plus particulièrement, l'invention concerne une molécule d'acide nucléique isolée qui code un polypeptide humain appelé récepteur 5 du facteur de nécrose tumorale, parfois appelé 'TNFR-5' ou 'TR5', ci-après dénommé 'récepteur TRAIL sans domaine intracellulaire' ou 'TRID'. L'invention concerne aussi des polypeptides TRID, des vecteurs, des cellules hôtes, et des méthodes de recombinaison pour les produire. Elle concerne enfin des méthodes de sélection permettant d'identifier des agonistes ou des antagonistes de l'activité du polypeptide TRAIL, ainsi que des méthodes diagnostiques et thérapeutiques mettant en oeuvre lesdites compositions.
PCT/US1998/000152 1997-01-14 1998-01-13 Recepteur 5 du facteur de necrose tumorale WO1998030693A2 (fr)

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DE69834027T DE69834027D1 (de) 1997-01-14 1998-01-13 Tumor-nekrose-faktor rezeptor 5
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US7049402B2 (en) 2006-05-23
ATE321855T1 (de) 2006-04-15
JP2001505060A (ja) 2001-04-17
US6261801B1 (en) 2001-07-17
EP0990031B1 (fr) 2006-03-29
AU6238698A (en) 1998-08-03
WO1998030693A9 (fr) 1999-09-02
WO1998030693A3 (fr) 1998-08-13
EP0990031A2 (fr) 2000-04-05
US20010021516A1 (en) 2001-09-13
DE69834027D1 (de) 2006-05-18

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